EP0912739A2 - Compositions de proteine ob canine et procedes - Google Patents

Compositions de proteine ob canine et procedes

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Publication number
EP0912739A2
EP0912739A2 EP97915853A EP97915853A EP0912739A2 EP 0912739 A2 EP0912739 A2 EP 0912739A2 EP 97915853 A EP97915853 A EP 97915853A EP 97915853 A EP97915853 A EP 97915853A EP 0912739 A2 EP0912739 A2 EP 0912739A2
Authority
EP
European Patent Office
Prior art keywords
protein
leu
canine
ser
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97915853A
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German (de)
English (en)
Inventor
Natasha Hernday
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Amgen Inc
Original Assignee
Amgen Inc
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Filing date
Publication date
Application filed by Amgen Inc filed Critical Amgen Inc
Publication of EP0912739A2 publication Critical patent/EP0912739A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to canine OB protein and related nucleic acids, vectors, host cells, methods of production, selective binding molecules, derivatives, pharmaceutical compositions, and diagnostic, therapeutic and cosmetic methods.
  • OB gene OB gene
  • protein protein encoded
  • the OB protein is active in vivo in both ob/ob mutant mice (mice obese due to a defect in the production of the OB gene product) as well as in normal, wild type mice.
  • the biological activity manifests itself in, among other things, weight loss. See generally. Barinaga, "Obese” Protein Slims Mice, Science 269: 475-476 (1995) .
  • Obesity may cause cardiovascular problems incident to high blood lipid levels, artherial plaque, high cholesterol, and high blood pressure.
  • Type II diabetes is associated with obesity. All these conditions may be present in dogs, particularly dogs fed diets with inappropriate levels of protein or fat.
  • the present invention provides for canine OB protein and associated nucleic acids, vectors, host cells, processes for production, related compositions, and methods for making and using thereof.
  • FIGURE 1 Presented is an alignment of the amino acid sequences for the present canine OB protein (top sequence, referred to as a pre-protein in reference to the presence of a leader sequence) and native human OB protein (Zhang et al., supra) (bottom, also referred to as a pre-protein) .
  • top sequence referred to as a pre-protein in reference to the presence of a leader sequence
  • native human OB protein Zhang et al., supra
  • FIGURE 2 Presented is an alignment of the amino acid sequences for the present canine OB protein (top sequence, again referred to as a pre-protein) and native murine OB protein (Zhang et al., supra) (bottom, also referred to as a pre-protein) .
  • top sequence again referred to as a pre-protein
  • native murine OB protein Zhang et al., supra
  • bottom also referred to as a pre-protein
  • FIGURE 3 Presented is an alignment of OB protein amino acid sequences of canine, human, murine and a consensus amino acid sequence.
  • the consensus sequence listed is based on conserved amino acid regions. Amino acids at positions which differ between species are assigned an amino acid on the basis of which amino acid is most prevalent.
  • the present invention provides for canine OB protein and associated nucleic acids, vectors, host cells, processes for production, related compositions, and methods for making and using thereof.
  • the full length canine OB protein is presented in Seq. ID. No. 1 (below) .
  • the leader sequence is (with respect to the numbering of Seq. ID No. 1) -21 to -1.
  • the mature protein consists essentially of (with respect to the numbering of Seq. ID No. 1) 1 to 146. Also, the present canine OB protein may have present or absent the glutamine ("Q") present at position +28 (with respect to the mature protein numbering of Seq.ID No. 1) .
  • Q glutamine
  • the present canine OB protein may be selected from among the amino acid sequences (with respect to Seq. ID No. 1) :
  • the present canine OB protein may optionally be formulated with a pharmaceutically acceptable diluent, adjuvant, or carrier.
  • Novel nucleic acid sequences of the invention include sequences useful in securing expression in procaryotic or eucaryotic host cells of canine OB protein selected from among the amino acid sequences (with respect to Seq. ID No. 1) :
  • DNA sequences of the invention specifically comprise: (a) the DNA of Seq. ID No. 2 (below); (b) the portion of the DNA sequence of Seq. ID No. 2 (below) encoding amino acids 1 through 146.
  • DNA sequences encoding allelic variant forms of canine OB protein such as those forms encoding the amino acid sequence with the gluta ine at position 28 absent, and manufactured DNA sequences encoding canine OB protein.
  • Such manufactured sequences may readily be constructed according to the methods of Alton et al., PCT published application WO 83/04053.
  • Genomic DNA encoding the present canine OB protein may contain additional non-coding bases, or introns, and such genomic DNAs are obtainable by hybridizing all or part of the cDNA, illustrated in Seq. ID NO. 2 , to a genomic DNA source, such as a canine genomic DNA library.
  • genomic DNA will encode functional canine OB protein; however, use of the cDNAs may be more practicable in that, since only the coding region is involved, recombinant manipulation is facilitated.
  • the present nucleic acid sequences may include the incorporation of codons "preferred" for expression by selected nonmammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences which facilitate construction of readily expressed vectors.
  • antisense nucleic acids against the present DNAs may be prepared. Such antisense nucleic acids may be useful in modulating the effects of OB protein in vivo. For example, one may prepare an antisense nucleic acid which effectively disables the ability of a cell to produce canine OB protein.
  • DNA sequences of the invention are also suitable materials for use as labeled probes in isolating human genomic DNA encoding canine OB protein, as mentioned above, and related proteins as well as cDNA and genomic DNA sequences of other mammalian species.
  • DNA sequences may also be useful in various alternative methods of protein synthesis (e.g., in insect cells) or in genetic therapy.
  • DNA sequences of the invention are expected to be useful in developing transgenic mammalian species which may serve as eucaryotic "hosts" for production of and products in quantity. See, generally, Palmiter et al., Science 221- 809-814 (1983) .
  • DNA sequences described herein which encode canine OB protein are valuable for the information which they provide concerning the amino acid sequence of the mammalian protein which have heretofore been unavailable.
  • DNA sequences provided by the invention are useful in generating new and useful viral and circular plasmid DNA vectors, new and useful transformed and transfected procaryotic and eucaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture) , and new and useful methods for cultured growth of such host cells capable of expression of canine OB protein.
  • RNAs may also be used for gene therapy for, example, treatment of conditions characterized by the insufficient expression of OB protein, such as obesity.
  • vectors suitable for gene therapy such as retroviral or adenoviral vectors modified for gene therapy purposes and of purity and pharmaceutical acceptability
  • Such vectors may incorporate nucleic acid encoding the present polypeptides for expression in a desired location.
  • Gene therapy may involve a vector containing more than one gene for a desired protein.
  • nucleic acid may be placed within a pharmaceutically acceptable carrier to facilitate cellular uptake, such as a lipid solution carrier (e.g., a charged lipid), a liposome, or polypeptide carrier (e.g., polylysine) .
  • a pharmaceutically acceptable carrier such as a lipid solution carrier (e.g., a charged lipid), a liposome, or polypeptide carrier (e.g., polylysine) .
  • lipid solution carrier e.g., a charged lipid
  • liposome e.g., a liposome
  • polypeptide carrier e.g., polylysine
  • Such methods for production include culturing, under suitable conditions, a host cell containing DNA encoding canine OB protein.
  • a host cell containing DNA encoding canine OB protein Provided herein is an in vitro population of such host cells.
  • Such host cell may be eukaryotic or prokayotic, and may contain a vector including the canine OB encoding DNA as provided herein.
  • canine OB protein DNA-containing host cell may have been modified via homologous recombination (see e.g., U.S. Patent No. 5,272,071 and WO 91/09955 as mentioned supra) or other methods for altered expression of canine OB protein.
  • one may further purify or process such canine OB protein, and prepare desired formulations.
  • transgenic dogs having altered canine OB levels. This may be accomplished by preparation of transgenic dogs.
  • One way to prepare a transgenic dog is to genetically manipulate a dog embryo with a "transgene", which typically includes a promoter and a desired DNA.
  • the desired DNA may be selected from those enumerated above.
  • the promoter may be for tissue specific expression, such as liver specific expression.
  • One or more of such embryos is then implanted into a pseudo-pregnant foster mother dog. The embryo develops and the dog gives birth.
  • One skilled in the art will recognize other means of preparing transgenic animals.
  • Such animals may be transgenic for increased canine OB protein expression, thus making leaner animals, or transgenic to "knock-out" canine OB expression via, for example, using a defective canine OB DNA as a transgene.
  • One or more of such animals may be prepared at a time (e.g., multiple embryo implants and multiple births); and such animals may be further bred.
  • transgenic dogs having altered canine OB protein expression. Such alteration may be in terms of amount (i.e., increased or decreased) or location of canine OB protein expression (i.e., tissue specific expression) .
  • progeny of such transgenic animals Such animals may be for pets, for show dogs, for breeding purposes, or for providing n vivo bioassays. Derivatives. Pharmaceutical Compositions
  • the present protein may also be derivatized by the attachment of one or more chemical moieties to the protein moiety.
  • the chemically modified derivatives may be further formulated for intraarterial, intraperitoneal, intramuscular, subcutaneous, intravenous, oral, nasal, pulmonary, topical or other routes of administration.
  • Chemical modification of biologically active proteins has been found to provide additional advantages under certain circumstances, such as increasing the stability and circulation time of the therapeutic protein and decreasing immunogenicity. See U.S. Patent No. 4,179,337, Davis et al., issued December 18, 1979. For a review, see Abuchowski et al., in
  • the chemical moieties suitable for derivatization may be selected from among various water soluble polymers.
  • the polymer selected should be water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer will be pharmaceutically acceptable.
  • One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/protein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations.
  • the - in ⁇ effectiveness of the derivatization may be ascertained by administering the protein or derivative, in the desired form (i.e., by osmotic pump, or, more preferably, by injection or infusion, or, further formulated for oral, pulmonary or nasal delivery, for example) , and observing biological effects as described herein.
  • the water soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random or non-random copolymers), and dextran or poly (n-vinyl pyrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols, polystyrenemaleate and polyvinyl alcohol.
  • Polyethylene glycol propionaldenhyde may have advantages in manufacturing due to its stability in water.
  • the desired molecular weight of the polymer may be determined empirically, based on desired characteristics, as well as the degree of
  • Fusion proteins may be prepared by attaching polyaminoacids to the OB protein moiety.
  • the polyamino acid may be a carrier protein which serves to increase the circulation half life of the protein.
  • such polyamino acid should be those which have do not create neutralizing antigenic response, or other adverse response.
  • Such polyamino acid may be selected from the group consisting of serum album (such as canine serum albumin), an antibody or portion thereof (such as an antibody constant region, sometimes called "F c ”) or other polyamino acids.
  • the location of attachment of the polyamino acid may be at the N-terminus of the OB protein moiety, or other place, and also may be connected by a chemical "linker" moiety, such as a polyamino acid linker, to the OB protein.
  • polymer molecules so attached may vary, and one skilled in the art will be able to ascertain the effect on function.
  • One may mono-derivatize, or may provide for a di-, tri-, tetra- or some combination of derivatization, with the same or different chemical moieties (e.g., polymers, such as different weights of polyethylene glycols) .
  • the proportion of polymer molecules to protein (or peptide) molecules will vary, as will their concentrations in the reaction mixture.
  • the optimum ratio in terms of efficiency of reaction in that there is no excess unreacted protein or polymer
  • the desired degree of derivatization e.g., mono, di-, tri-, etc.
  • the molecular weight of the polymer selected whether the polymer is branched or unbranched, and the reaction conditions.
  • the chemical moieties should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
  • attachment methods available to those skilled in the art.
  • EP 0 401 384 herein incorporated by reference (coupling PEG to G-CSF) , see also Malik et al. , Exp. Hematol. 2£: 1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride) .
  • a reactive group such as, a free amino or carboxyl group may be used.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residue.
  • Those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydrl groups may also be used as a reactive group. Attachment at residues important for receptor binding should be avoided if receptor binding is desired.
  • N-terminally chemically modified protein One may specifically desire N-terminally chemically modified protein. Selective N-terminal chemical modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved. For example, one may selectively N-terminally pegylate the protein by performing the reaction at a pH which allows one to take advantage of the pK a differences between the e-amino group of the lysine residues and that of the a-amino group of the N-terminal residue of the protein.
  • the water soluble polymer may be of the type described above, and should have a single reactive aldehyde for coupling to the protein.
  • N-terminally monopegylated derivative is preferred for ease in production of a therapeutic.
  • N-terminal pegylation ensures a homogenous product as characterization of the product is simplified relative to di-, tri- or other multi pegylated products.
  • the use of the above reductive alkylation process for preparation of an N-terminal product is preferred for ease in commercial manufacturing.
  • Pharmaceutical Compositions In yet another aspect of the present invention, provided are methods of using pharmaceutical compositions of the proteins, and derivatives. Such pharmaceutical compositions may be for administration by injection, or for oral, pulmonary, nasal, transdermal or other forms of administration.
  • compositions comprising effective amounts of protein or derivative products of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite) , preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol) ; incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes.
  • buffer content e.g., Tris-HCl, acetate, phosphate
  • additives e.g., Tween 80, Polysorbate 80
  • anti-oxidants e.g., ascorbic acid, sodium metabisulfite
  • preservatives e.g., Thimersol,
  • Hylauronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
  • Such compositions may influence the physical state, stability, rate of in vivo release, and rate of n vivo clearance of the present proteins and derivatives. ⁇ ej ⁇ , e.g. , Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference.
  • the compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form.
  • Implantable sustained release formulations are also contemplated, as are transdermal formulations. Contemplated for use herein are oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences, 18th Ed.
  • Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets.
  • liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673) .
  • Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (E.g., U.S. Patent No. 5,013,556) .
  • a description of possible solid dosage forms for the therapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and CT. Rhodes Chapter 10, 1979, herein incorporated by reference.
  • the formulation will include the protein (or analog or derivative) , and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
  • Protein may be chemically modified so that oral delivery of the derivative is efficacious.
  • the chemical modification contemplated is the attachment of at least one moiety to the protein (or peptide) molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine.
  • the increase in overall stability of the protein and increase in circulation time in the body examples include: Polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
  • One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the protein (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.
  • a coating impermeable to at least pH 5.0 is essential.
  • examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate
  • CAT hydroxypropylmethylcellulose phthalate
  • HPMCP 50 HPMCP 55
  • PVAP polyvinyl acetate phthalate
  • Eudragit L30D Eudragit L30D
  • Aquateric cellulose acetate phthalate
  • CAP Eudragit L
  • Eudragit S Eudragit S
  • Shellac Shellac
  • a coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow.
  • Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used.
  • the shell material of cachets could be thick starch or other edible paper.
  • moist massing techniques can be used.
  • the therapeutic can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1mm.
  • the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
  • the therapeutic could be prepared by compression.
  • the protein may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a food or drink.
  • diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
  • Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
  • Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
  • Disintegrants may be included in the formulation of the therapeutic into a solid dosage form.
  • Materials used as disintegrates include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
  • Another form of the disintegrants are the insoluble cationic exchange resins.
  • Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
  • Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC) , ethyl cellulose (EC) and carboxymethyl cellulose (CMC) . Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
  • MC methyl cellulose
  • EC ethyl cellulose
  • CMC carboxymethyl cellulose
  • PVP Polyvinyl pyrrolidone
  • HPMC hydroxypropylmethyl cellulose
  • Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE) , liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added.
  • the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • a surfactant might be added as a wetting agent.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride.
  • nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose.
  • surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.
  • Additives which potentially enhance uptake of the protein (or derivative) are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
  • Controlled release formulation may be desirable.
  • the drug could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms i.e. gums.
  • Slowly degenerating matrices may also be incorporated into the formulation.
  • Another form of a controlled release of this therapeutic is by a method based on the Oros therapeutic system (Alza Corp.), i.e. the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects.
  • Some entric coatings also have a delayed release effect.
  • Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan.
  • the therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups.
  • the first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols.
  • the second group consists of the enteric materials that are commonly esters of phthalic acid.
  • Film coating may be carried out in a pan coater or in a fiuidized bed or by compression coating.
  • pulmonary delivery of the present protein, or derivative thereof is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
  • the formulation of the molecule will be such that between about .10 ⁇ g/kg/day and lg/kg/day will yield the desired therapeutic effect.
  • the effective dosages may be determined using diagnostic tools over time. For example, a diagnostic for measuring the amount of OB protein in the blood (or plasma or serum) may first be used to determine endogenous levels of OB protein. Such diagnostic tool may be in the form of an antibody assay, such as an antibody sandwich assay. The amount of endogenous OB protein is quantified initially, and a baseline is determined.
  • the therapeutic dosages are determined as the quantification of endogenous and exogenous OB protein (that is, protein, analog or derivative found within the body, either self-produced or administered) is continued over the course of therapy.
  • the dosages may therefore vary over the course of therapy, with a relatively high dosage being used initially, until therapeutic benefit is seen, and lower dosages used to maintain the therapeutic benefits.
  • a further embodiment of the invention is selective binding molecules, such as monoclonal antibodies selectively binding canine OB protein.
  • the hybridoma technique described originally by Kohler and Milstein Eur. J. Immunol. £, 511-519 (1976) has been widely applied to produce hybrid cell lines that secrete high levels of monoclonal antibodies against many specific antigens.
  • Recombinant antibodies (see Huse et al., Science 246: 1275 (1989)) may also be prepared.
  • Such recombinant antibodies may be further modified, such as by modification of complementarity determining regions to increase or alter affinity, or alteration of constant regions to resemble canine constant regions of such antibodies.
  • Such antibodies may be incorporated into a kit for diagnostic purposes, for example.
  • a diagnostic kit may be employed to determine the location and/or amount of canine OB protein of an individual. Diagnostic kits, such as for a "sandwich" -type assay, may also be used to determine if an individual has receptors which bind canine OB protein, or those which, to varying degrees, have reduced binding capacity or ability. As stated infra, such antibodies may be prepared using immunogenic portions of a canine OB protein, particularly the immunogenic portions which are unique to canine OB protein.
  • Therapeutic uses include weight reduction, the treatment or prevention of diabetes, blood lipid reduction (and treatment of related conditions) , increasing lean body mass and increasing insulin sensitivity.
  • the present compositions may be used for manufacture of one or more medicaments for treatment or amelioration of the above conditions.
  • Weight Reduction The present compositions and methods may be used for weight reduction. As has been demonstrated in murine models (see supra) , administration of the present OB protein results in weight loss.
  • the weight loss is primarily of adipose tissue, or fat. Such weight loss can be associated with the treatment of concomitant conditions, such as those below, and therefore constitute a therapeutic application.
  • cosmetic uses are provided herein if weight loss is solely for improvement in appearance, such as for show dogs or dogs used for breeding.
  • the present compositions and methods may be used in the prevention or treatment of Type II diabetes.
  • Type II diabetes can be correlated with obesity
  • use of the present invention to reduce weight can also alleviate or prevent the development of diabetes.
  • the present compositions may be used to prevent or ameliorate diabetes.
  • the present compositions and methods may be used in the modulation of blood lipid levels. Ideally, in situations where solely reduction in blood lipid levels is desired, or where maintenance of reduction of blood lipid levels is desired, the dosage will be insufficient to result in weight loss. Thus, during an initial course of therapy of an obese animal, dosages may be administered whereby weight loss and concomitant blood lipid level lowering is achieved. Once sufficient weight loss is achieved, a dosage sufficient to prevent re-gaining weight, yet sufficient to maintain desired blood lipid levels may be administered. These dosages can be determined empirically, as the effects of OB protein are reversible. E.g.. Campfield et al., Science 269: 546-549 (1995) at 547. Thus, if a dosage resulting in weight loss is observed when weight loss is not desired, one would administer a lower dose in order to achieve the desired blood lipid levels, yet maintain the desired weight.
  • Lean mass increase without weight loss may be achieved sufficient to decrease the amount of insulin (or, potentially, amylin or other potential diabetes treating drugs) an individual would be administered for the treatment of diabetes.
  • Lean mass increase with concomitant increase in overall strength may be achieved with doses insufficient to result in weight loss.
  • Other benefits, such as an increase in red blood cells (and oxygenation in the blood) and a decrease in bone resorption or osteoporosis may also be achieved in the absence of weight loss.
  • compositions and methods may be used in conjunction with other therapies, such as altered diet and exercise.
  • Other medicaments such as those useful for the treatment of diabetes (e.g., insulin, and possibly amylin) , cholesterol and blood pressure lowering medicaments (such as those which reduce blood lipid levels or other cardiovascular medicaments) , activity increasing medicaments (e.g., amphetamines), and appetite suppressants.
  • Such administration may be simultaneous or may be in seriatim.
  • the present methods may be used in conjunction with surgical procedures, such as cosmetic surgeries designed to alter the overall appearance of a body (e.g., liposuction or laser surgeries designed to reduce body mass, or implant surgeries designed to increase the appearance of body mass) .
  • cardiac surgeries such as bypass surgeries or other surgeries designed to relieve a deleterious condition caused by blockage of blood vessels by fatty deposits, such as arterial plaque
  • Methods to eliminate gall stones such as ultrasonic or laser methods, may also be used either prior to, during or after a course of the present therapeutic methods.
  • the present methods may be used as an adjunct to surgeries or therapies for broken bones, damaged muscle, or other therapies which would be improved by an increase in lean tissue mass.
  • RNAs or DNAs may be used to characterize or detect defects in an individual's canine OB DNA gene or gene product.
  • an obese individual may have a defective canine OB gene.
  • the present DNAs may be used to hybridize with the nucleic acid from an individual to detect such defects, such as via PCR techniques.
  • Canine OB protein may be used to characterize an individual's OB protein for its ability to bind to OB receptor, or for other biological activity. For example, one may prepare an assay for the ability of OB protein to alter lipid metabolism by preparing a population of lipid containing cells expressing the OB receptor, and contacting OB protein with such population of cells.
  • Modulation of lipid content, characteristics of lipid or other characteristics may be monitored.
  • the present protein or nucleic acids may be associated with a detectable labels substance such as a radioactive isotope, a fluorescent or chemiluminescent chemical, or other label available to one skilled in the art.
  • a detectable labels substance such as a radioactive isotope, a fluorescent or chemiluminescent chemical, or other label available to one skilled in the art.
  • Such nucleic acids may be used for tissue distribution assays (for example, to detect the distribution of canine OB mRNA transcripts in different tissue types) or for other assays to determine the location of OB receptor.
  • the first amino acid of the mature protein is at position +1 and is a valine ("V") and is indicated in bold print.
  • V valine
  • recognition sites for agents which cleave amino acids for ease in production of protein without the N-terminal methionyl residue.
  • one may include an enzymatic recognition site at the N-terminus for ease in production (abbreviated as "1-146 met -1") .
  • DNA may alter the DNA (Seq. ID. No. 2) yet not alter the amino acid sequence which is encoded.
  • Such alterations may be, for example, to include restriction sites for ease in replication or vector insertion, or to include codons preferred for expression in certain systems, such as bacterial expression or eukaryotic expression.
  • Canine OB Protein (Seg. ID No. 1)

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Abstract

Cette invention se rapporte à la protéine OB canine et à des compositions et des procédés associés. Des séquences d'acides aminés OB canins, des séquences d'acides nucléiques associées, des vecteurs, des cellules hôtes, des procédés de production par recombinaison, des anticorps associés et/ou des compositions diagnostiques, des compositions pharmaceutiques et des animaux transgéniques sont présentés.
EP97915853A 1996-03-01 1997-02-28 Compositions de proteine ob canine et procedes Withdrawn EP0912739A2 (fr)

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US6001968A (en) * 1994-08-17 1999-12-14 The Rockefeller University OB polypeptides, modified forms and compositions
US6001816A (en) * 1996-06-20 1999-12-14 Merck & Co., Inc. Gene therapy for leptin deficiency
EP0954579A1 (fr) * 1996-06-20 1999-11-10 Merck & Co., Inc. Therapie genique pour le traitement de l'obesite
EP0950417A3 (fr) 1998-02-23 2000-02-23 Pfizer Products Inc. Traitement d' anomalies du squelette
AU3863699A (en) * 1998-04-20 1999-11-08 Mayo Foundation For Medical Education And Research Treatment of osteoporosis with leptin
SE9904424D0 (sv) * 1999-07-13 1999-12-03 Sahltech Ab Use of interleukin-6 in combination with leptin in treatment of obesity
US10105447B2 (en) * 2013-03-13 2018-10-23 Elanco Us Inc. Method of treating obesity in a companion animal comprising administering a modified canine leptin polypeptide
EP3481413A4 (fr) * 2016-07-08 2020-01-08 Askgene Pharma, Inc. Protéine de fusion comprenant de la leptine et procédés de production et d'utilisation associés

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US6309853B1 (en) * 1994-08-17 2001-10-30 The Rockfeller University Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof
FI973162L (fi) * 1995-01-31 1997-09-30 Lilly Co Eli Liikalihavuutta vastustavia proteiineja
CA2221824A1 (fr) * 1995-05-26 1996-11-28 Eli Lilly And Company Proteine et adn de gene ob de rhesus

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WO1997032022A3 (fr) 1997-10-09
JP2000513564A (ja) 2000-10-17
KR19990087392A (ko) 1999-12-27
HUP0000280A2 (hu) 2000-06-28
AU2317397A (en) 1997-09-16
CA2247503A1 (fr) 1997-09-04
US20020142456A1 (en) 2002-10-03
HUP0000280A3 (en) 2002-09-30

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